nicholas f. dybdal-hargreaves texas health science center ......10 center at san antonio, san...

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Eribulin Mesylate: Mechanism of Action of a Unique Microtubule-Targeting 1 Agent 2

Nicholas F. Dybdal-Hargreaves1, April L. Risinger1,2, and Susan L. Mooberry1,2 3 4 1Department of Pharmacology, University of Texas Health Science Center at San 5 Antonio, San Antonio, Texas; 2Cancer Therapy & Research Center, University of 6 Texas Health Science Center at San Antonio, San Antonio, Texas 7 8 Corresponding Author: Susan L. Mooberry, University of Texas Health Science 9 Center at San Antonio, San Antonio, 7703 Floyd Curl Drive, Mail Code 7764, San 10 Antonio, TX 78229-3900. Phone: 210-567-4788; Fax 210-567-4300; E-mail: 11 [email protected] 12 13 Running Title: Eribulin: A Unique Microtubule-Targeting Agent 14 15 Disclosure of Potential Conflicts of Interest 16 S.L. Mooberry reports receiving commercial research support from Eisai Inc., 17 speakers bureau honoraria from CMEducation Resources, and is a 18 consultant/advisory board member for Eisai Inc. No potential conflicts of interest 19 were disclosed by the other authors. 20

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Abstract 1 Eribulin mesylate (eribulin), an analog of the marine natural product halichondrin 2 B, is a microtubule-depolymerizing drug that has utility in the treatment of 3 patients with breast cancer. Clinical trial results have demonstrated that eribulin 4 treatment provides a survival advantage to patients with metastatic or locally 5 advanced breast cancer previously treated with an anthracycline and a taxane. 6 Furthermore, a pooled analysis of two pivotal phase III trials has demonstrated 7 that eribulin also improves overall survival in several patient subgroups, including 8 in women with human epidermal growth factor receptor 2 (HER2)-negative 9 disease and triple-negative breast cancer. This review covers the preclinical 10 research that led to the clinical testing and approval of eribulin, as well as 11 subsequent research that was prompted by distinct and unexpected effects of 12 eribulin in the clinic. Initial studies with halichondrin B, and then eribulin, 13 demonstrated unique effects on tubulin binding that resulted in distinct 14 microtubule-dependent events and antitumor actions. Consistent with the actions 15 of the natural product, eribulin has potent microtubule-depolymerizing activities 16 and properties that distinguish it from other microtubule targeting agents. Here, 17 we review new results that further differentiate the effects of eribulin from other 18 agents on peripheral nerves, angiogenesis, vascular remodeling and epithelial-19 to-mesenchymal transition. Together, these data highlight the distinct properties 20 of eribulin and begin to delineate the mechanisms behind the increased survival 21 benefit provided by eribulin for patients. 22 23




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Introduction 1 Eribulin mesylate (eribulin) is a novel microtubule targeting agent (MTA) that is 2 used in the treatment of metastatic breast cancer. The EMBRACE clinical trial 3 showed a survival advantage for pretreated patients with locally recurrent or 4 metastatic breast cancer who were treated with eribulin when compared with 5 treatment of physician’s choice (1). These positive results contributed to the 6 approval of eribulin (Halaven®, Eisai Inc., Woodcliff Lake, NJ, USA) in the USA in 7 2010, and in Europe and Japan in 2011. A pooled analysis of the EMBRACE and 8 Study 301 phase III trials has also shown that eribulin improves overall survival in 9 several patient subgroups with advanced/metastatic breast cancer who have 10 previously received an anthracycline and a taxane. Among those who may 11 benefit from eribulin are women with HER2 negative and triple-negative breast 12 cancer (2). 13

This review describes the initial interest in the halichondrins as potential 14 anticancer drugs and the preclinical development of eribulin, including initial 15 establishment of the biochemical and cellular mechanisms of its microtubule 16 destabilizing activity. In addition, we describe new in vitro and in vivo studies 17 triggered by unexpected clinical findings that establish a distinct biological profile 18 of eribulin’s effects in both tumor tissue and supporting stroma. Ultimately, this 19 review seeks to highlight the differences in the biological effects of eribulin in 20 comparison with other MTAs that might contribute to its clinical activities. 21 From Halichondrin B to Eribulin 22




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Halichondrin B (Fig. 1), was isolated in 1986 from the sponge Halichondria 1 okadai based on its cytotoxicity (3). Halichondrin B was found to have 2 extraordinary cytotoxic potency in vitro and antitumor activity against murine 3 models of solid tumors and leukemia in vivo; however, only low yields of the 4 compound could be obtained (4). Evaluation of halichondrin B in the NCI-60 cell 5 line panel using the COMPARE algorithm revealed a pattern similar to that of 6 other tubulin binding agents, suggesting a microtubule-dependent mechanism of 7 action (4). Detailed mechanistic studies showed that halichondrin B inhibited 8 tubulin polymerization and tubulin-dependent guanosine triphosphate (GTP) 9 hydrolysis in a manner distinct from that of other microtubule destabilizers (4). 10

Halichondrin B was found to bind within a region of tubulin designated the 11 ‘vinca domain’, at which drugs such as dolastatin 10 bind and non-competitively 12 inhibit the binding of vinca alkaloids (4, 5). The binding properties of halichondrin 13 B were similar to those of dolastatin 10; however, there were notable differences 14 between the compounds, in that halichondrin B caused distinct conformational 15 effects on tubulin (6). 16

Halichondrin B was therefore mechanistically interesting, with promising 17 antitumor effects, but its complex structure and the low yield from natural sources 18 severely limited its potential for clinical development. A breakthrough occurred in 19 1992, when the Kishi laboratory succeeded in the total synthesis of halichondrin 20 B (7). This allowed the design, synthesis and evaluation of many analogs of the 21 compound, one of which, eribulin (ER-086526, E7389, NSC-707389), is the 22 subject of this review. The story of how the daunting 63-step chemical synthesis 23




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of eribulin was developed and made economically feasible was recently 1 described (8). 2 Preclinical Studies on Eribulin 3 Identification of eribulin as a microtubule targeting agent 4 The promising biological effects of halichondrin B led to the synthesis of over 180 5 halichondrin B analogs (9, 10). Optimal preclinical activities were observed for 6 two macrocyclic ketone analogs, and after extensive preclinical testing, the C35 7 primary amine-substituted compound (eribulin; Fig. 1) was selected for clinical 8 development. 9

Towle et al. showed that eribulin had potent antiproliferative effects across 10 a panel of eight human cancer cells lines, with an average half maximal inhibitory 11 concentration (IC50) value of 1.8 nM (11). The cytotoxic effects of eribulin were 12 selective for proliferating cells, because no cytotoxicity was observed against 13 fully quiescent immortalized human fibroblasts at concentrations up to 1 µM (11). 14 Similar to other MTAs, eribulin caused cells to accumulate in mitosis with 15 aberrant mitotic spindles leading to apoptosis, suggesting that eribulin, like its 16 parent halichondrin B, was a MTA (11-13). In contrast to most other MTAs, the 17 mitotic blockade induced by eribulin was shown to be irreversible; this may be an 18 important feature that enables even transient drug exposure to result in long-term 19 loss of cell viability (13). The initial direct interaction of eribulin with tubulin was 20 also demonstrated (11). 21

Based on a combination of experimental and modeling data, it has been 22 proposed that eribulin binds within a pocket underneath the H3 and H11 loops of 23




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β-tubulin in a distinct manner from that of other MTAs (14). Studies with 1 radiolabeled eribulin and tubulin heterodimers demonstrated relatively low affinity 2 (46 μM) and 1:1 binding (15); however, eribulin binds with high affinity (3.5 μM) to 3 polymerized microtubules in vitro, in a concentration-dependent manner reaching 4 an extrapolated maximum of 14.7 molecules per microtubule: this is consistent 5 with the hypothesis that eribulin acts as a microtubule end poison by binding with 6 high affinity to each of the 13 β-tubulin subunits at the plus end of each 7 microtubule protofilament. 8 Effects of eribulin on microtubule dynamics 9 Using live traces of single microtubules made from purified bovine tubulin, Jordan 10 et al. discovered that eribulin promoted pausing of microtubule growth, and 11 strongly inhibited the growth rate of microtubules (16). However, unlike 12 vinblastine, eribulin had little to no effect on the microtubule-shortening rate. 13 Similar results were observed in live MCF7 interphase cells expressing green 14 fluorescent protein (GFP)-tubulin, in which 1 nM eribulin suppressed microtubule 15 growth with little effect on disassembly, and decreased microtubule dynamicity 16 overall (16). Eribulin also induced mitotic accumulation with aberrant mitotic 17 spindle dynamics at 1 nM, demonstrating an ability to impact overall microtubule 18 function at this concentration. Together, these results showed that eribulin 19 disrupted microtubule dynamics in a manner somewhat distinct from that of 20 vinblastine (17). The effects of eribulin on centromere dynamics during mitosis 21 were also evaluated (18). Live U2OS cells expressing GFP-labeled centromere 22 protein B, a component of the centromere, were used to measure functional 23




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microtubule dynamicity during mitosis. While control cells exhibited centromere 1 dynamics of 0.84 μm/min, 60 nM eribulin decreased centromere dynamics to 2 0.55 μm/min. In contrast to other MTAs, eribulin did not affect the mean 3 centromere separation distance, consistent with biochemical results indicating 4 that eribulin disrupts microtubules differently. 5 Effects of eribulin in murine xenograft models 6 Eribulin demonstrated a wide spectrum of antitumor activity in human xenograft 7 models of breast cancer, colon cancer, fibrosarcoma, glioblastoma, head and 8 neck cancer, leiomyosarcoma, ovarian cancer, melanoma, pancreatic cancer, 9 non-small-cell lung cancer and small-cell lung cancer (11, 19). In addition, 10 eribulin also had activity in cells from a range of pediatric solid tumors and acute 11 lymphocytic leukemia in vitro and in xenograft models (20). Collectively, these 12 preclinical studies established eribulin as a promising compound, that retained 13 the unique properties of halichondrin B and had excellent preclinical in vivo 14 activity. Importantly, eribulin also had an acceptable toxicity profile and 15 therapeutic window in mice across several dosing schedules. 16 Clinical Observations and Resulting Studies 17 The EMBRACE trial compared eribulin with treatment of physician’s choice, and 18 showed an overall survival advantage in patients treated with eribulin (1), kindling 19 new research to identify which properties of the drug might contribute to this 20 survival advantage. In addition, while the overall side effect profile was similar 21 between eribulin and the physician’s choice arm (which included other tubulin-22 targeting agents such as vinorelbine and several non-MTA therapies), there were 23




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also some differences. This led to further preclinical research, as described 1 below. 2 Peripheral neuropathy 3 Peripheral neuropathy is a well-documented toxicity of MTAs that can lead to 4 treatment discontinuation or dose reduction. Observations during the clinical 5 evaluation of eribulin indicated that the incidence and severity of peripheral 6 neuropathy might differ between eribulin and other MTAs (21). These initial 7 observations prompted comparative in vitro studies on the effects of eribulin, 8 ixabepilone, and paclitaxel in mice to determine differences in the effects of these 9 agents on peripheral nerves. 10

MTA-induced peripheral neuropathy in mice has been measured using 11 behavioral assays and causes altered sensitivity to painful stimuli (22). These 12 behavioral changes, indicative of peripheral neuropathy, are associated with 13 diminished peripheral nerve conduction velocity, amplitude and morphology. 14 After defining the individual maximum tolerated doses (MTDs) in mice, MTAs 15 were tested for their effects on parameters associated with peripheral neuropathy 16 in caudal and digital nerves at fractional doses (0.25, 0.5, 0.75 and 1) of the MTD 17 on a 2-week schedule (23). Eribulin did not change the nerve conduction velocity 18 of either nerve type, but did increase the caudal nerve amplitude at the MTD and 19 0.75 MTD. In contrast, at the MTD and 0.75 MTD, ixabepilone and paclitaxel 20 significantly decreased both nerve conduction velocity and amplitude. All three 21 drugs caused dose-dependent pathologies to L4 and L5 dorsal root ganglia and 22 sciatic nerves, including axonal degeneration, cytoplasmic vacuolation and dark 23




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inclusions (Fig. 2). Pathological changes occurred with each drug, but paclitaxel 1 and ixabepilone initiated more severe changes in sciatic nerves than did eribulin. 2 Tissue histology of the sciatic nerve showed that eribulin-induced pathologies 3 were less frequent and less severe than those observed with the other agents. 4 These investigators next studied whether eribulin exacerbated pre-existing 5 peripheral neuropathy in mice (24). Peripheral neuropathy was induced with a 2-6 week cycle of 0.75 MTD paclitaxel. After a 2-week recovery, the mice were 7 switched to either 0.5 MTD eribulin or 0.5 MTD paclitaxel for another 2 weeks. 8 Nerve conduction velocity and amplitude measurements demonstrated that 9 eribulin did not significantly worsen the existing nerve function; however, the 10 second treatment with paclitaxel did exacerbate the pre-existing neuropathy. 11 Nevertheless, both eribulin and paclitaxel treatments caused an increase in the 12 number of degenerated axons in the sciatic nerve as compared with pre-existing 13 neuropathy, suggesting that both drugs caused some degree of damage. 14

It has been noted that the peripheral neuropathy induced by MTAs initially 15 affects the longest nerves of the body, those innervating the feet and hands (25). 16 The susceptibility of these long neurons to MTAs suggests disruption of essential 17 microtubule-dependent transport. The effects of eribulin and other MTAs on 18 microtubule-dependent trafficking, anterograde and retrograde transport in squid 19 axoplasm were evaluated (26). Ixabepilone and vincristine both caused a 27% 20 decrease in anterograde trafficking and a ~20% decrease in retrograde trafficking 21 at 1 μM. In contrast, eribulin and paclitaxel caused less than a 20% decrease in 22 anterograde trafficking at concentrations of up to 10 μM, with no significant 23




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difference in retrograde transport. Importantly, there were no gross changes in 1 axoplasm microtubule structures under any of these conditions. Consistent with 2 the axoplasm data, experiments with purified biochemical components 3 demonstrated a direct inhibition of kinesin function with 10 μM ixabepilone or 4 vincristine, but not with eribulin or paclitaxel. These mechanistic studies, using a 5 variety of models, support the idea that eribulin has distinct effects on peripheral 6 nerves and are consistent with clinical results demonstrating that eribulin has a 7 low frequency of treatment discontinuation due to peripheral neuropathy (1, 27, 8 28). 9 Angiogenesis and vascular effects 10 Angiogenesis is necessary for tumor growth and metastasis, and is an important 11 drug target. Angiogenesis is complex, involving not only endothelial cells, but 12 also stromal pericytes that support and stabilize endothelial cells (29, 30). 13

The effects of eribulin on endothelial cells or pericytes alone or in co-14 culture were compared with the effects of paclitaxel (31). The proliferation of 15 human umbilical endothelial cells was potently inhibited by both eribulin and 16 paclitaxel, with similar IC50 values of 0.54 nM and 0.41 nM, respectively. 17 Interestingly, human brain vascular pericytes (HBVPs) were less sensitive to both 18 drugs, with IC50 values of 1.19 nM for eribulin and 2.19 nM for paclitaxel. Gene 19 expression analyses revealed that eribulin and paclitaxel altered similar genes in 20 endothelial cells (59% overlap; most with decreased expression levels), but had 21 distinct profiles in pericytes. In the HBVPs, only 12% of gene expression changes 22 overlapped between eribulin and paclitaxel treatment. Eribulin caused a general 23




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decrease in transcription, while paclitaxel caused an increase. Of the genes 1 affected by both drugs, 22 were down-regulated by eribulin but up-regulated by 2 paclitaxel. These data indicate that, compared with endothelial cells, HBVPs 3 respond differently to MTA treatment, and that eribulin and paclitaxel cause 4 divergent effects on pericyte gene expression. 5

To study the effects of eribulin and paclitaxel on capillaries, endothelial 6 cells and pericytes were co-cultured. Resultant capillary networks were 7 insensitive to high concentrations (1 µM) of eribulin or paclitaxel over 2 days, but 8 after 3 days of treatment eribulin initiated a loss of the capillary networks with an 9 IC50 of 3.6 nM. These networks remained resistant to paclitaxel until day 5, when 10 the IC50 for capillary loss was 13 nM. In the absence of pericytes, endothelial cell 11 tubules (formed in a collagen gel) were equally sensitive to inhibition by eribulin 12 or paclitaxel after 4 days, demonstrating the critical role played by pericytes in 13 establishing differential sensitivity between MTAs. These results highlight 14 differences between eribulin and paclitaxel with regard to their overall 15 antiangiogenic and antivascular activities, suggesting that they might affect these 16 processes differently in vivo, and that the basis for such differences may reside in 17 differential sensitivities of pericytes to the two agents. 18 Effects of eribulin on vascular remodeling 19 Tumor vasculature is very different from normal vasculature, with tortuous, 20 abnormal vessels that are targets for anticancer therapies. The abnormal tumor 21 vasculature, together with tumor and stromal proliferation, results in a high 22 interstitial tumor pressure, further impeding tumor perfusion and contributing to 23




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the hypoxic tumor environment (32). Tumor hypoxia and the accompanying 1 acidity are implicated in tumor progression, metastasis and drug resistance. 2 Several microtubule depolymerizing agents cause rapid vascular changes 3 associated with destruction of intratumoral vasculature, leading to a rapid 4 shutdown of tumor perfusion, and tumor necrosis (33-35). 5

Considering the antivascular effects observed with microtubule 6 depolymerizing agents, it was important to evaluate the effects of eribulin on 7 tumor perfusion. Dynamic contrast enhanced magnetic resonance imaging 8 (DCE-MRI) experiments in the nude rat MX-1 and MDA-MB-231 human 9 xenograft models of breast cancer showed that 0.3 mg/kg doses of eribulin 10 caused an increase in the tumor perfusion transfer coefficient (Ktrans), indicative of 11 increased perfusion in the tumor core. Representative DCE-MRI images of the 12 MX-1 tumors before and after eribulin treatment are shown in Fig. 3, together 13 with the average Ktrans measurements of the tumor rim and core (36). Similar 14 results were obtained in a murine xenograft model, using Hoechst 33342 dye to 15 measure perfusion (31). Tumors also showed an increase in the number of 16 microvessels after eribulin treatment, consistent with increased perfusion. These 17 data collectively demonstrate that eribulin causes tumor vascular remodeling, 18 leading to increased perfusion. 19

Mechanistic studies showed that eribulin decreased the expression of 20 genes associated with angiogenesis. This included genes involved in the 21 vascular endothelial growth factor (VEGF), Wnt, Notch and ephrin signaling 22 pathways, and in the epithelial-to-mesenchymal transition (EMT), a process 23




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known to be driven in part by hypoxia (37). At the protein level, eribulin 1 decreased the expression of both VEGF and CA9, markers of hypoxia. 2

An increase in tumor perfusion would be expected to alleviate the hypoxic 3 environment of the tumor. This could improve subsequent systemic 4 chemotherapy, both by reducing hypoxia-driven chemoresistance and by 5 enhancing intratumoral delivery of drugs. This hypothesis was tested using 6 capecitabine with and without eribulin pretreatment. Strikingly, eribulin-pretreated 7 tumors were significantly more sensitive to subsequent capecitabine treatment 8 than non-pretreated tumors, consistent with the hypothesis that improved 9 perfusion due to eribulin treatment could increase the cytotoxic efficacy of a 10 subsequent treatment. Thus, eribulin induces tumor vasculature remodeling, 11 leading to increased perfusion of the tumor, which can result in diminished 12 hypoxia and enhanced antitumor efficacy of subsequent therapies. It is 13 interesting to speculate that such vascular changes, at this time only observed in 14 preclinical models, might contribute to the clinical efficacy and survival advantage 15 observed in the EMBRACE trial. 16 Epithelial-to-mesenchymal transition 17 Similar to angiogenesis, EMT is considered a crucial process in tumor 18 progression and metastasis. During EMT, the gene signature and phenotype of 19 epithelial cells change in such a way that they adopt mesenchymal 20 characteristics that have been implicated in increased drug resistance, enhanced 21 invasion and metastasis, and a shift toward stem cell phenotypes (38). Drugs 22 with the ability to inhibit or to reverse EMT are highly desired. Due to the survival 23




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advantage that eribulin demonstrated clinically, it was hypothesized that eribulin 1 may affect EMT. Notably, treatment with 1 nM eribulin for 1 week in three 2 mesenchymal-like triple-negative breast cancer cell lines triggered reversal of 3 EMT in surviving cells as suggested by increased epithelial-like morphology and 4 significant changes in gene profiles and protein marker expression (39). The 5 surviving cells showed increased messenger RNA (mRNA) expression of the 6 epithelial markers E-cadherin and keratin 18, as well as decreased expression of 7 multiple mesenchymal markers including N-cadherin, vimentin, TWIST and ZEB-8 1. 9

Changes in the protein levels of E-cadherin, N-cadherin and vimentin in 10 surviving eribulin-treated cells mirrored the changes in mRNA levels (39). 11 Consistent with these effects, fully viable, surviving MX-1 cells showed 12 significantly diminished capacities for in vitro migration and invasion. In addition, 13 eribulin also caused similar EMT reversal in MX-1 tumor xenografts in vivo (39). 14 Consistent with the direct effects on cells in vitro, eribulin caused a shift of 15 residual tumors to a more epithelial phenotype, with increased E-cadherin and 16 lower tumor expression of N-cadherin and ZEB-1 (Fig. 4). To evaluate the 17 metastatic propensity of drug-treated cells, MX-1 cells were treated in vitro with 18 vehicle, eribulin or 5-fluorouracil (5-FU), and equal numbers of surviving cells 19 were injected into the tail veins of mice. Treatment with eribulin, but not with 5-20 FU, strongly inhibited lung nodule formation 15 days after injection of the cells, 21 and 60% of the mice survived for 80 days following the injection. In contrast, all 22 of the mice injected with vehicle or 5-FU-pretreated cells died within 21 days 23




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following injection. While these results are exciting, further studies are necessary 1 to determine if this contributes to the survival advantage of eribulin. 2 Concluding Remarks 3 Many different assay systems measuring diverse parameters collectively show 4 that eribulin has many characteristics that make it distinct from other MTAs. 5 Although still speculative, the specific biochemical effects of eribulin may underlie 6 its unique effects on peripheral nerves, angiogenesis, vascular remodeling and 7 EMT. Studies continue to demonstrate that while different MTAs have many 8 shared mechanisms of action, they also clearly have non-overlapping effects. 9 Recent evidence by several researchers has highlighted the importance of 10 microtubules in normal interphase events, suggesting that the clinical distinctions 11 between MTAs could be due, at least in part, to their differential effects on 12 interphase microtubules (40). Additionally, there is evidence that eribulin has 13 profound effects on the tumor microenvironment that differ from those of 14 paclitaxel and possibly other MTAs. These mechanistic studies have the potential 15 to improve and guide the use of MTAs in specific tumor types and in 16 mechanistically driven drug combinations. It is likely that ongoing research into 17 these features of MTAs will reveal many of the underlying reasons for the 18 efficacy of these agents in cancer therapy, and show how they can best be used 19 to improve patient survival. 20 Acknowledgments 21




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The authors extend sincere thanks to Dr. Bruce A. Littlefield (Eisai Inc., Andover, 1 MA, USA) for his insights and contributions to this review. Editorial support was 2 provided by Oxford PharmaGenesis and was funded by Eisai Inc. 3 4




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37. Jung HY, Fattet L, Yang J. Molecular pathways: linking tumor 1 microenvironment to epithelial-mesenchymal transition in metastasis. Clin 2 Cancer Res 2015;21:962-8. 3 38. Steinestel K, Eder S, Schrader AJ, Steinestel J. Clinical significance of 4 epithelial-mesenchymal transition. Clin Transl Med 2014;3:17. 5 39. Yoshida T, Ozawa Y, Kimura T, Sato Y, Kuznetsov G, Xu S, et al. Eribulin 6 mesilate suppresses experimental metastasis of breast cancer cells by reversing 7 phenotype from epithelial-mesenchymal transition (EMT) to mesenchymal-8 epithelial transition (MET) states. Br J Cancer 2014;110:1497-505. 9 40. Komlodi-Pasztor E, Sackett DL, Fojo AT. Inhibitors targeting mitosis: tales 10 of how great drugs against a promising target were brought down by a flawed 11 rationale. Clin Cancer Res 2012;18:51-63. 12 13




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Figure 1. Chemical structures of halichondrin B and eribulin. 1 Figure 2. Effect of MTDs of eribulin mesylate, paclitaxel and ixabepilone on 2 sciatic nerve morphology. Tissue from mice treated with A, vehicle, B, eribulin, C, 3 paclitaxel or D, ixabepilone. Severe pathological changes consistent with axonal 4 degeneration of both large and small fibers are highlighted with red arrows and 5 white arrowheads, respectively. No regeneration (e.g. thin myelinated fibers) was 6 evident with paclitaxel or ixabepilone. Scale bar, 20 µm. MTD, maximum 7 tolerated dose. 8 Reprinted from ref. 23: Cancer Research 2011;71(11):3952–62, Wozniak KM, 9 Nomoto K, Lapidus RG, Wu Y, Carozzi V, Cavaletti G, et al. Comparison of 10 neuropathy-inducing effects of eribulin mesylate, paclitaxel, and ixabepilone in 11 mice, with permission from AACR. 12 Figure 3. Eribulin increases tumor vascular perfusion in an MX-1 rat xenograft 13 model of human breast cancer. A. Representative DCE-MRI images of MX-1 14 tumors showing initial area under the curve (iAUC) maps prior to (pretreatment 15 day-1) or on day 6 following eribulin treatment (0.1 or 0.3 mg/kg, days 0 and 4). A 16 difference in perfusion between the tumor rim and tumor core is seen prior to 17 treatment as indicated by lighter colors on the tumor rim and darker colors in the 18 core. Changes in tumor perfusion after treatment are indicated by the change in 19 color of the tumor core. B. Average volume transfer constant values (Ktrans) in 20 tumor rim or tumor core regions as determined by DC-MRI. *P < 0.05 versus 21 vehicle. VEH, vehicle; ERI, eribulin; 0.1, 0.3, dose in mg/kg, every fourth day 22 beginning in day 0; iAUC, initial area under the curve (36). 23




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Reprinted from ref. 36 with permission of Blackwell Publishing Ltd.: Cancer 1 Science. Funahashi Y, Okamoto K, Adachi Y, Semba T, Uesugi M, Ozawa Y, et 2 al. Eribulin mesylate reduces tumor microenvironment abnormality by vascular 3 remodeling in preclinical human breast cancer models. Cancer Sci 4 2014;105:1334–42. Copyright 2014 The Authors. 5 Figure 4. Eribulin reverses EMT in MX-1 human breast cancer xenografts in 6 vivo. A, Schematic representation of treatment scheme. B, Representative 7 immunohistochemistry images of human (i.e. tumor, not host) E-cadherin 8 (upper), N-cadherin (middle) and ZEB-1 (lower) in tumor specimens from animals 9 treated with eribulin 0.3, 1 and 3 mg/kg. Images taken at 100 × magnification. C, 10 Quantification of immunohistochemistry staining of the markers shown in B. Data 11 for individual tumors are presented as points, with mean ± standard error of the 12 mean of the group shown by horizontal lines and error bars (n = 10). ***P < 13 0.001, ****P < 0.0001 versus control group (Dunnett-type multiple comparison 14 test). EMT, epithelial-to-mesenchymal transition; IHC, immunohistochemistry; 15 WB, Western blotting; ZEB-1, zinc finger E-box-binding homeobox 1. 16 Reprinted from ref. 39 by permission from Nature Publishing Group and 17 Macmillan Publishers Ltd on behalf of Cancer Research UK: British Journal of 18 Cancer (http://www.nature.com/bjc/index.html). Yoshida T, Ozawa Y, Kimura T, 19 Sato Y, Kuznetsov G, Xu S, et al. Eribulin mesilate suppresses experimental 20 metastasis of breast cancer cells by reversing phenotype from epithelial-21 mesenchymal transition (EMT) to mesenchymal-epithelial transition (MET) 22 states. Br J Cancer 2014;110:1497-505. Copyright 2014. 23




© 2015 American Association for Cancer Research

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Published OnlineFirst April 2, 2015.Clin Cancer Res Nicholas F. Dybdal-Hargreaves, April L Risinger and Susan L Mooberry Microtubule-Targeting AgentEribulin Mesylate: Mechanism of Action of a Unique

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